Development of a NIR Iridium(III) Complex-Based Probe for the Selective Detection of Iron(II) Ions

Abstract

As a commonly used metal ion, iron(II) (Fe²⁺) ions pose a potential threat to ecosystems and human health. Therefore, it is particularly important to develop analytical techniques for the rapid and accurate detection of Fe²⁺ ions. However, the development of near-infrared (NIR) luminescence probes with good photostability for Fe²⁺ ions remain challenging. In this work, we report a novel iridium(III) complex-based luminescence probe for the sensitive and rapid detection of Fe²⁺ ions in a solution based on an Fe²⁺-mediated reduction reaction. This probe is capable of sensitively detecting Fe²⁺ ions with a limit of detection (LOD) of 0.26 μM. Furthermore, this probe shows high photostability, and its luminescence remains stable under 365 nm irradiation over a time period of 30 min. To our knowledge, this is first iridium(III) complex-based NIR probe for the detection of Fe²⁺ ions. We believe that this work provides a new method for the detection of Fe²⁺ ions and has great potential for future applications in water quality testing and human monitoring.

1. Introduction

Iron (Fe) is the second most abundant element in the Earth’s crust and is also an essential nutrient in the human body [1]. In fact, approximately half of the iron in the human body is in the form of Fe²⁺ ions and Fe³⁺ [2], which are involved in many important physiological processes in the human body [3], such as oxygen transfer [4], nucleotide synthesis [5], electron transfer [6], and enzymatic reactions [7]. However, the presence of excess redox-active free Fe catalyzes the generation of hydroxide anions and increases the production of reactive oxygen species (ROS) [8,9], and abnormal levels of Fe²⁺ ions are highly associated with diseases including cancer and inflammation [10]. Therefore, the amount of water-soluble fraction of Fe²⁺ ions in the body is an important indicator for estimating Fe-induced cellular damage. On the other hand, the release of large amounts of Fe-containing chemicals into the environment may lead to a range of environmental and ecological problems [11]. Therefore, the monitoring and detection of Fe²⁺ ion levels are crucial in protecting environmental and living systems.

Traditional analytical techniques have been widely developed for the detection of Fe²⁺ and Fe³⁺ ions, such as colorimetric [12,13], electrochemical [14], and fluorescent methods [15,16]. Due to the fact that Fe²⁺ ions are easily oxygenated to Fe³⁺ ions in aerobic water [17], and numerous biometallic ions can be competitive in binding to common ligands, it is still a challenge to develop probes for the highly selective detection of Fe²⁺ ions. Reaction-based fluorescence probes offer great potential in overcoming the limitations above, which combine Fe²⁺-mediated reactions and the advantages of fluorescent techniques, including high sensitivity, high specificity, simple operation, and a fast response time [18]. In particular, the Fe²⁺-mediated N-oxide reduction strategy, first reported by Nagasawa et al. [19], has been demonstrated as a reliable and selective method to construct Fe²⁺ ion fluorescence probes. However, most of these probes suffer from narrow Stokes shifts, poor photostability, or a short emission wavelength of below 650 nm.

Recently, NIR iridium(III) complexes have emerged as promising luminescence probes for chemosensing and bioimaging [20] due to their long emission lifetime, high photostability, large Stokes shift, and tunable emissions. Moreover, NIR iridium(III) complex-based probes are highly resistant to background interferences and provide more accurate detection results [21]. These merits offer the possibility of compensating the shortcomings of the traditional Fe²⁺ ion fluorescence probes. Although a considerable number of NIR iridium(III) complexes have been developed for the detection of metal ions, small molecules, and disease-related proteins [22,23,24,25,26,27], no NIR iridium(III) complex-based probes are available for the detection of Fe²⁺ ions.

In this work, inspired by the Fe²⁺-mediated N-oxide reduction reaction, we rationally designed and synthesized a NIR iridium(III) complex for the detection of Fe²⁺ ions in an aqueous solution (Scheme 1). Complex 1 showed a quenching luminescence upon the addition of Fe²⁺ ions through the Fe²⁺-mediated N-oxide moiety reduction of the N^N ligand. Moreover, the complex exhibited a good performance, with high selectivity and high sensitivity for the detection of Fe²⁺ ions, and a fast response of within 3 min.

2. Results and Discussion

2.1. Design and Synthesis of the Probe for Fe²⁺ Ions

Fe²⁺ is a common divalent cation that can reduce oxides to their original elements or compounds [28]. This inspired us to design an oxide-containing NIR iridium(III) complex, in which the N^N ligand containing the N-oxide group was responsible for the specific recognition of Fe²⁺ ions, while 6,7-difluoro-2-methyl-3-phenylquinoxaline was chosen to act as C^N ligand to enable NIR emission (Scheme 1). It was expected that Fe²⁺ ions would trigger deoxygenation to generate complex S3 and induce a luminescence quenching effect on complex 1. Moreover, the weak complexation of Fe²⁺ ions with the O atom of the N-oxide might also promote the break of the N–O bond [29]. The complex was directly synthesized in four steps (Scheme 2): 1,10-phenanthroline-5,6-dione and N,N-diethylaminobenzaldehyde were cyclized under the catalyst of ammonium acetate (NH₄OAc) to generate ligand S1, which was then substituted with lodomethane (CH₃I) in the presence of potassium tert-butoxide (t-BuOK) to produce ligand S2. Ligand S2 coordinated with the chloro-bridged iridium(III) dimer Ir₂(dfpq)₄Cl₂ for the generation of complex S3, which was further oxidized by 3-chloroperoxybenzoic acid (m-CPBA) to afford the desired complex 1. The purity of complex 1 was checked by high-performance liquid chromatography (HPLC), which showed a purity of over 95% (Figure S9). The intermediates and final complex were fully characterized by ¹H NMR, ¹³C NMR, and electrospray ionization mass spectrometry (ESI-MS) (Figures S1–S8).

2.2. Photophysical Properties of the Probe

The absorption spectrum of complex 1 was first recorded in dimethyl sulfoxide (DMSO), which showed the assumed ligand-based π−π* transition peak at around 280 nm and a metal-to-ligand charge transfer (MLCT) transition peak at around 365 nm and 480 nm in DMSO (Figure 1a). The excitation and emission spectra showed that complex 1 had the maximum excitation wavelength of 365 nm and the maximum wavelength of 675 nm (Figure 1b and Figure S10). This indicates that complex 1 is a potential NIR probe and has a large Stokes shift of around 195 nm between the ³MLCT absorption and emission, which is much larger than organic dyes [30], making it less interfered with by the excitation light source. In order to assess the photostability, complex 1 was continuously irradiated over a time period of 1800 s at 365 nm (Figure 1c), showing no obvious decrease in luminescence intensity, which confirms its high photostability, ensuring reliable reproducibility and stability in the experimental results.

Following the encouraging outcomes delineated above, we assessed the capacity of complex 1 in detecting Fe²⁺ ions across various solvent media. Complex 1 (10 μM) was added to various solutions, including ACN, ethanol (EtOH), N,N-dimethylformamide (DMF), DMSO, and acetone containing Fe²⁺ ions (50 μM), respectively; luminescence spectra showed that the best quenching effect was obtained in DMSO (Figure 1d), mainly due to cyclometalated iridium(III) complexes being lipophilic and having better solubility in DMSO and being slight soluble in alcohols [31,32]. In summary, complex 1 has the merits of a large Stokes shift, good photostability, NIR emission, and the best response to Fe²⁺ ions in DMSO, which is better than that for Fe²⁺ ion probes with an emission below 600 nm (Table S1).

2.3. Optimization of Detection Conditions

In pursuit of optimal detection performance, a systematic optimization of both the incubation time and the concentration of the complex was conducted. Firstly, the luminescence response of complex 1 (20 μM) to Fe²⁺ (50 μM) over a time period of 30 min was investigated; the results showed that the luminescence dramatically dropped, reaching a plateau at 3 min (Figure 2a). Notably, this rapid response time contrasts with the behavior exhibited by several previously reported fluorescent probes (Table S1), which typically necessitate incubation periods of around 30 min or longer to achieve a plateau. Further, the luminescence response of complex 1 at different concentrations (5–20 μM) to Fe²⁺ (50 μM) within 3 min showed that the highest quenching effect was obtained at 20 μM (Figure 2b). Therefore, 3 min of incubation time and 20 μM of complex 1 were chosen for further experiments.

2.4. Sensitivity and Selectivity of Complex 1

After optimizing the reaction conditions, we investigated the luminescence response of complex 1 to varying concentrations of Fe²⁺ ions (0–50 μM). It is found that the luminescence of complex 1 decreased gradually with increasing concentrations of Fe²⁺ ions (Figure 3a, with a good linear relationship obtained between 0.5 and 20 μM (Figure 3b, Inset), obtaining a limit of detection (LOD) of 0.26 μM with a regression equation of Y = −0.038X + 0.8293 according to LOD = 3σ/k, and a sensitivity (S_i) of −0.038. This is comparable to most of the reported fluorescence methods and better than the reported colorimetric methods (Table S1).

In order to assess the selectivity of complex 1 towards potentially interfering metal ions, the luminescence response of complex 1 (20 μM) to a range of metal ions in DMSO was investigated. The results demonstrated that the luminescent behavior of complex 1 remained consistent upon the introduction of other metal ions (at 10 equiv. concentrations, encompassing Li⁺, Na⁺, K⁺, Ag⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Ba²⁺, Al³⁺, Fe³⁺, and Au³⁺ ions. Conversely, the subsequent addition of Fe²⁺ ions (50 μM) led to a substantial quenching of the luminescent signal emitted by the probe (Figure 3c). This indicates that the co-existence of other metal ions exerts a negligible influence over the luminescence of the complex toward Fe²⁺ ions, confirming its excellent selectivity. This attribute confers a distinct advantage over coordination-based Fe²⁺ ion probes (Table S1), as it minimizes the susceptibility to interference from various other metal ions.

2.5. Mechanism Study

To validate the response mechanism of complex 1 for the detection of Fe²⁺ ions, ESI-MS was used to confirm the reduction of the N-oxide group by Fe²⁺ ions to afford control complex S3. The ESI-MS spectrum shows that the presence of Fe²⁺ ions induced a new m/z peak at 1084.30, corresponding to that of complex S3, while the m/z peak of complex 1 is 1100.74 (Figure 4). In addition, the reaction mechanism was further characterized by HPLC, and the result shows that the presence of Fe²⁺ ions induced the emergence of a peak with a residual time of around 18.60 min, compared with the residual time of about 14.00 min for complex 1 (Figure S11), which is consistent with complex 1 being more polar than complex S3. These results demonstrate that Fe²⁺ ions can reduce complex 1 to S3, and the reduction of the N-oxide group to an amino group presumably induces a photoinduced electron transfer (PeT) between the amino group and the iridium(III) complex, which transfers electrons from the donor to the excited state of the iridium(III) complex [33], leading to a strong luminescence quenching effect in complex 1.

2.6. The Recovery by Complex 1 of Fe²⁺ Ions in Water Samples

The contamination associated with Fe²⁺ ions poses a significant threat to human health, making the practical application of complex 1 in environmental samples highly important. In this study, the feasibility of complex 1 in detecting Fe²⁺ ions in environment samples was investigated using water from 2% Xi’an Qixiang Lake as a model matrix. Complex 1 (20 μM) was mixed with Fe²⁺ ions at varying concentrations (5, 10, and 20 μM) in 2% lake-water-spiked DMSO, the recovery rates were found to range from 89.71% to 111.07%, with RSD values of 2.85% to 6.34% (Table S2). This result demonstrates the analytical potential of complex 1 in quantifying Fe²⁺ ions in environmental matrices.

3. Conclusions

In conclusion, we have successfully designed and synthesized a novel NIR luminescence probe for Fe²⁺ ion detection in aqueous solution. The complex can respond rapidly to Fe²⁺ ions within 3 min, exhibiting a high sensitivity, with an LOD of 0.26 μM. Moreover, the complex shows high selectivity for Fe²⁺ ions over other common metal ions, even in the presence of other interferents, and demonstrates a high photostability, with the luminescence remaining similar even under UV irradiation for 30 min. A further mechanism study demonstrates that the complex detects Fe²⁺ ions through Fe²⁺ ion-mediated N-oxide reduction, triggering a PeT process, along with luminescence quenching. In summary, this work provides a new analytical tool for the sensitive and selective detection of Fe²⁺ ions in aqueous solution, opening up a new avenue for the robust detection of Fe²⁺ ions. In future, we anticipate that leveraging advanced nanotechnological methodologies will facilitate the further development of this probe into a versatile array of tools [34]; these tools may encompass live imaging applications for the detection of Fe²⁺ ions within cancer systems, the targeted analysis of Fe²⁺ ions in cancerous tissues, and precision analyses dedicated to Fe²⁺ ion quantification in both untreated environmental specimens and blood samples.